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IntroductionDespite the 100 or so years that have lapsed since French

scientist Louis-Camille Maillard first reported the Maillard reaction between amino acids and glucose in 1912, in vivo research has not progressed significantly. In recent years, advances have been made in proteomics research, which is the comprehensive analysis of expressed protein, thanks in large part to the human genome project. However, this research subsequently led to the identification of diseases which cannot be cured simply by studying gene or protein expression. The Maillard reaction has also recently attracted new attention for its role as a posttranslational modification, promoted by an abnormality in the metabolism of sugars and lipids. Advanced glycation end products (AGEs) change the physicochemical properties of proteins such as net negative chargedegeneration and polymerization.

Recent studies have reported that formation of AGEs is enhanced by not only abnormalities in the metabolism of sugar but also by elevated oxidative stress. Further, the interaction between AGEs and receptor for AGEs (RAGE) also cause of inflammation and oxidative stress. Here, we describe the biological significance of AGEs and RAGE as risk factors for aging.

Review Article

Advanced Glycation End Products and Their Receptors as Risk Factors for Aging

Ryoji Nagai 1), Masao Jinno 2), Masamitsu Ichihashi 3), Hidenori Koyama 4), Yasuhiko Yamamoto 5), Yoshikazu Yonei 3)

1) Laboratory of Food and Regulation Biology Department of Bioscience, School of Agriculture, Tokai University2) Women’s Clinic Jinno3) Anti-Aging Medical Research Center, Graduate School of Life and Medical Sciences, Doshisha University4) Department of Endocrinology and Metabolism, Hyougo Medical College5) Department of Biochemistry and Molecular Vascular Biology, Kanazawa University Graduate School of Medical Science

AbstractGlycation is the reaction between amino residues of proteins and carbonyl of reducing sugars. French Louis Camille Maillard

discovers this reaction from the browning reaction by amino acid and sugar, and it is widely known for the food chemistry as the Maillard reaction. The hemoglobin A1c (HbA1c) measured all over the world as a marker of glycemic control is equivalent to the Amadori rearrangement products, which is the early stage product of the Maillard reaction. Therefore, glycation progresses even in the healthy subject since carbohydrates are used as an energy source. Then, the Amadori products are changed to Advanced Glycation End-products (AGEs), which shows the autofluorescence, browning, and cross-linking by oxidation, dehydration, and a condensation reaction. Furthermore, AGE-proteins are recognized by AGE receptor such as RAGE (receptor for AGE). This review describes the proposed Pathways for the formation of AGEs during the Maillard reaction and role of the AGEs in the pathogenesis of age-related diseases.

KEY WORDS: AGEs, glycation, RAGE

Received: Dec 13, 2011 Accepted: May. 15, 2012Published online: Jun. 30, 2012

Anti-Aging Medicine 9 (4) : 108-113, 2012(c) Japanese Society of Anti-Aging Medicine

Ryoji NagaiLaboratory of Food and Regulation Biology Department of Bioscience, School of Agriculture, Tokai University

Kawayou, Minamiaso, Aso-gun, Kumamoto 869-1404, JapanTel & Fax: +81-967-67-3918 / E-mail: nagai-883@umin.ac.jp

Maillard reaction (glycation reaction)Taking its name from its discoverer, the Maillard reaction

is also known as glycation, as it progresses from the reaction in which reducing sugar combines with protein. The reaction can be divided into two parts: the first half proceeds until Amadori rearrangement, and the second half involves AGE formation through reactions such as oxidation, dehydration, and condensation (Fig. 1). Food chemists have conducted substantial research on this reaction since its initial discovery in relation to food processing, browning by storage, and change in nutritive value.

Hemoglobin A1c (HbA1c) was first identified in vivo in the 1970s, and subsequent research has revealed this molecule to be the Amadori rearrangement product of glucose combining with the N-terminal amino residue of valine on the hemoglobin β chain. While HbA1c is used globally as a marker for diabetic glycemic control, clinical application of AGEs is limited by the large number of AGE structures and the fact that measuring methods differ by structure. However, as carbohydrates are nutrients indispensable to energy production, an organism uses these compounds throughout its life, and Maillard reactions therefore progress as long as it is alive. Positively charged lysine and arginine residues on proteins are modified by AGEs, and increased net negative charge of proteins and forming cross-linkage structures are also known to influence the structure of proteins, including the activity of enzymes (Fig. 2).

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Fig. 1. Maillard reaction.

Fig. 2. Generation of intermediate aldehydes and protein degeneration involved in AGE formation.

In the past, measurement of f luorescence intensity has primarily involved estimating the AGE content, which is a relatively simple process. However, a number of concerns have been raised regarding estimation of AGEs in vivo simply by measuring f luorescence intensity, given the great number of AGEs which do not fluoresce and the fact that some non-AGE compounds show similar f luorescence properties to AGEs, potentially confounding results.

In light of these drawbacks, assessment of AGE structures based on anti-AGE antibodies has been widely employed, given the inherent advantage with its multi-sample measurement capabilities. In vivo AGE research has rapidly progressed as well,

revealing that AGE generation in living organisms involves not only glucose but also various carbonyl compounds formed by metabolism and inflammation reactions (Fig. 3), and that several formation pathways exist for short-term rapid production of AGEs 1,2). In recent years, a substantial amount of focus has been directed towards the study of AGEs in the field of aging research, as AGE formation is promoted in patients with lifestyle-related diseases accelerated by aging, such as metabolic disorders involving carbohydrates . Detailed measurements of certain antibodies and instrumental analyses of several AGEs have also accelerated the progress of research in this field.

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Receptors for AGEs (RAGE)Currently identified cell surface receptors which recognize

AGEs include RAGE; macrophage scavenger receptor class A (SR-A); 3) SR-B (SR-B1 and CD36); 4-5) lectin-like oxidized low density lipoprotein receptor-1 (LOX-1); 6) galectin-3 complex; 7) fasciclin , epidermal growth factor (EGF)-like, laminin-type EGF-like, and link domain-containing scavenger receptors-1 and -2 (FEEL-1 and -2); megalin; 8) and toll-like receptor 4 (TLR4) (Fig. 4). RAGE and TLR4, both pattern-recognition receptors (PRRs), are believed to be responsible for intracellular signal transduction. RAGE is a type 1 transmembrane protein belonging to the immunoglobulin superfamily and is known to be a multi-ligand receptor recognizing not only AGEs but also advanced oxidation protein products (AOPPs) resulting from oxidative stress 9), amyloid beta related to Alzheimer’s disease 10), high-mobility group box-1 (HMGB-1) linked to cancer metastasis and inflammation 11), inflammatory mediator S100 protein secreted from immune cells 12), Mac1/CD11b on the cell surface of white blood cells, lipopolysaccharide (LPS) of bacterial membrane components 13), complement C3a, and phosphatidylserine on apoptotic cells 14).

A typical intracellular signaling pathway of RAGE involves formation of intracellular oxidation stress and activation of transcription factor NFκB. Studies in diabetic transgenic mice overexpressing RAGE protein in vascular cells have shown that the onset of diabetic nephropathy is accelerated in this model. In contrast, studies in RAGE knockout mice with diabetes have shown that nephropathy was ameliorated in these animals 15). Taken together, these previous findings demonstrate that RAGE is functionally involved in the development of vascular complications in diabetes.

In addition, RAGE-dependent inflammatory disorders were demonstrated by animal models such as atherosclerosis, the

Fig. 3. Pathway of intermediate aldehyde generation.

pulmonary fibrosis caused by bleomycin exposure , osteoporosis, sepsis, Alzheimer’s disease and colitis. Endogenous secretory RAGE (esRAGE) is a secretory form of RAGE made by alternative splicing of the RAGE gene 16), and soluble RAGE (sRAGE) is formed by shedding full-length RAGE via activation of enzymes such as MMP9 or ADAM10. Both esRAGE and sRAGE have ligand-binding sites and work as decoy receptors by capturing RAGE ligands 17).

Current progress in AGE research A number of studies have reported the marked accumulation

of the AGE structure Nε-(carboxymethyl)lysine (CML) in the elastic fiber of solar elastosis 18), with related studies reporting a clear relationship between CML formation and reactive oxygen species (ROS) such as hydroxyl radicals 19), peroxynitrite 20), and hypochlorous acid 21). In solar elastosis, CML is believed to accumulate in elastic fibers in response to production of hydroxyl radicals by ultraviolet rays, prompting active research into the relationship between solar elastosis and AGEs.

While the relationship between f lecks and glycation is unclear at present, the association between wrinkle formation and glycation of collagen is considered to be one of the causes of elastin glycation and the cross-linking of elastic fiber. Exposure to ultraviolet rays induces the production of reactive oxygen species (ROS)—and thereby AGE formation. Prevention of dermal aging and solar elastosis will therefore require inhibiting not only glycation stress but also ROS production in response to skin exposure to ultraviolet A radiation.

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Recent research has focused on the relationship between AGEs and regenerative medical techniques. AGEs have been found to affect follicular growth, fertilization, embryonic development, and pregnancy outcome 22), and AGE accumulation is now believed to be a new index for rating ovarian dysfunction, independent from age and day-3-FSH and therefore quite useful for early diagnosis. In addition, reducing AGE accumulation may represent a new method for treating infertility.

Several reports, including those by our group, have recently demonstrated the presence of autoantibodies against AGEs in human plasma. Shibayama et al. reported that the titer of autoantibodies against CML in patients with diabetes was significantly higher than in normal subjects 23). Similarly, Mera et al. also found elevated levels of autoantibodies against several AGE structures in plasma from healthy human subjects, with titers of autoantibodies against Nε-(carboxyethyl)lysine (CEL) particularly high compared with those of other AGE structures such as CML and pentosidine (Fig. 5) 24). Further studies will be needed to clarify the pathophysiological role of autoantibodies against AGE structures and their potential to be used as biological markers or therapeutic targets.

Future ResearchAs described above, in vivo research on AGEs is not limited

only to studies on diabetic complications but has extended into various fields, consequently bringing to light points requiring

further investigation. However, despite the increasingly apparent importance of clarifying which AGE structures accumulate in which tissue types, as AGE formation pathways and structure are known to vary by pathological condition and tissue in which they accumulate, no studies have yet thoroughly examined AGE structures.

Clinical laboratories can measure AGE concentration in blood using anti-AGE antibodies via an immunochemical technique wherein serum samples are heated to 100 °C for 15 minutes to inactivate the protease. However, heating biological samples, as in foods, results artificial formation of AGEs such as CML 25) and pentosidine 26) via Amadori rearrangement.

Development of an effective inhibitor of AGE formation in anti-aging treatment will ultimately require (1) identifying the structures of target AGEs, (2) analyzing the formation pathways of target AGEs, and (3) developing a method of measuring AGEs that does not involve the formation of artificial AGEs.

Conflict of interest statement:This work was supported in part by Grants-in-Aid for

Scientific Research (No. 24300260 and 24650482 to Ryoji Nagai) from the Ministry of Education, Science, Sports and Cultures of Japan.

Fig. 4. Receptors for AGEs.

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Fig. 5. Levels of anti-AGE autoantibodies in healthy subjects.

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